Cryobiology最新文献

{"title":"Graphene oxide and silymarin combination: A novel approach to improving post-cryopreservation quality of ram sperm.","authors":"Mohsen Shayestehyekta, Mojtaba Moradi","doi":"10.1016/j.cryobiol.2025.105199","DOIUrl":"10.1016/j.cryobiol.2025.105199","url":null,"abstract":"<p><p>Graphene oxide (GO) has been extensively studied for its diverse biomedical applications, including drug delivery, imaging, and tissue engineering. Silymarin, as a flavonoid complex derived from the milk thistle plant, has recently shown potential health benefits, particularly concerning reproductive health. This study aims to evaluate the effects of GO and silymarin supplementation, both individually and in combination, on the characteristics of frozen-thawed ram sperm. Semen samples were collected using standard artificial insemination (AI) techniques with an artificial vagina. The collected semen was evaluated and cryopreserved in a tris-based extender containing varying concentrations of silymarin and GO (0, 10, or 20 μg/mL) or their combination. Post-thaw assessments evaluated sperm motility, viability, morphological abnormalities, DNA integrity, membrane integrity, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, and total antioxidant capacity (TAC). Our findings revealed that the combination of 20 μg/mL silymarin and 20 μg/mL GO significantly enhanced total motility, viability, membrane integrity, and DNA integrity of sperm. Additionally, this treatment effectively reduced morphological abnormalities and MDA levels post-thawing. Notably, SOD and TAC activities were improved following the freeze-thaw compared to other treatment groups. In conclusion, the combination of silymarin and GO significantly improves the quality of frozen-thawed ram sperm by enhancing sperm parameters while reducing oxidative stress markers. The results suggest their potential as effective additives in cryopreservation protocols, providing a promising avenue for improving reproductive outcomes in rams and potentially other livestock species.</p>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":" ","pages":"105199"},"PeriodicalIF":2.3,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142969880","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative and qualitative histological evaluation of different regions of cryopreserved skin derived from red-rumped agouti for obtaining cryobanks.","authors":"João Vitor da Silva Viana, Érika Almeida Praxedes, Luanna Lorenna Vieira Rodrigues, Yasmin Beatriz França Moura, Leonardo Vitorino Costa de Aquino, Moacir Franco de Oliveira, Alexsandra Fernandes Pereira","doi":"10.1016/j.cryobiol.2024.105190","DOIUrl":"10.1016/j.cryobiol.2024.105190","url":null,"abstract":"<p><p>Skin banks are valuable tools for the maintenance of biodiversity. The red-rumped agouti is a wild rodent of ecological importance in South America because it acts as a seed disperser, and skin banks could serve as alternatives to conserve genetic variability. Nevertheless, the most suitable skin region for forming these banks must still be determined to guarantee tissue quality after cryopreservation. We harvested skin tissues from the ear, abdominal, and inguinal regions of red-rumped agouti and evaluated the effects of cryopreservation on the quantitative and qualitative histological parameters of these somatic tissues. All tissues were evaluated for ultrastructure, skin thickness, cell count, number of perinuclear halos, collagen density, proliferative activity, and cellular ability during culture. Cryopreservation altered the thicknesses of the abdominal and inguinal dermises. Cryopreservation did not alter the number of fibroblasts and perinuclear halos in any region. The abdominal region had a higher number of fibroblasts than the other regions. All groups showed increased collagen fibers after cryopreservation, while maintaining a similar proliferative potential. During culture, all regions presented with cell viability above 90 % before and after cryopreservation, and the doubling time was maintained. However, cells from the inguinal and abdominal regions had lower metabolic rates than those from the ear. In summary, the ear skin was found to be the best region for cell recovery. However, abdominal skin has shown positive results and may be an alternative source of somatic cells.</p>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":" ","pages":"105190"},"PeriodicalIF":2.3,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142930572","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Effect of hydroxypropyl cellulose on vitrification of sheep embryos.","authors":"Quanbao Wang, YVting Ding, Jianhong Gao, Lijie Xu, Chang Liu, Xiaoyan Dong, Haixing Liu","doi":"10.1016/j.cryobiol.2024.105192","DOIUrl":"https://doi.org/10.1016/j.cryobiol.2024.105192","url":null,"abstract":"<p><p>Vitrification is a conventional and mature method for embryo cryo-preservation, but ice crystals formed during the vitrification process can damage embryos. HPC has the property of forming a high-viscosity gel under low-temperature conditions, so it can be added to vitrification solutions to investigate whether it improves the negative impact of vitrification on embryos. The results showed that the addition of HPC (50 μg/ml) to the vitrification solution significantly increased the post-warming survival rate of sheep morula embryos. Compared to the vitrification group, the addition of HPC significantly reduced the level of ROS (reactive oxygen species, ROS) and increased the level of GSH (glutathione, GSH) in embryos after warming, mitigating the damage to mitochondria caused by vitrification. It also had clear positive effects on the developmental potential of vitrified-warmed sheep embryos. Overall, the addition of 50 μg/ml HPC to the vitrification solution can significantly improve the quality of sheep morula embryos after vitrification and warming.</p>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"118 ","pages":"105192"},"PeriodicalIF":2.3,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142946112","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Probing the impact of commercial cryoprotectants and freezing technique on the motility of human spermatozoa cryopreserved onto extremely water-repellent soot-coated surfaces.","authors":"Karekin D Esmeryan, Yulian I Fedchenko, Todor A Chaushev","doi":"10.1016/j.cryobiol.2025.105195","DOIUrl":"10.1016/j.cryobiol.2025.105195","url":null,"abstract":"<p><p>The cryopreservation of human spermatozoa is an integral part of cryobiology, aiming to support the in-vitro fertilization. The latter relies on the availability of as much as possible reproductively active spermatozoa, whose number after thawing decreases due to the accompanied freezing injury and the cytotoxicity of cryoprotectants. An innovative option to circumvent these obstacles is to make the freezing interface non-wettable, by coating it with rapeseed oil soot possessing intrinsic cryoprotective properties, delaying the ice formation and possibly providing identical rates of intracellular dehydration and extracellular crystallization. It may mean that this technique can reduce or avoid the need of harmful cryoprotective agents, but to reach such a developmental stage, the synergistic effect of certified cryoprotectants and cooling velocities on the efficiency of soot-mediated sperm cryopreservation must be clarified. With the intention to address this research gap, we reveal that the slow freezing/thawing of distinct mixtures of three cryoprotectants (SpermFreeze™, CryoSperm™ and DMSO) and the human semen of nine patients equalizes the percent of survived spermatozoa, but declines their curvilinear velocity. At instant freezing (∼7-20 s) and slow thawing, via specially-designed soot fabric-coated sheet metal cryoboxes, the inclusion of 10 % DMSO is noxious, but the post-thaw motility reaches 74-100 % independently of the cryoprotective solutes. These surprising findings are ascribed to the formation of a quasivitrified semen, whose complete freezing ensures a fraction of extracellular ice matching that from the equilibrium phase diagram, eluding the osmotic shocks and paving the path for future replacement of the classic vitrification.</p>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":" ","pages":"105195"},"PeriodicalIF":2.3,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142930654","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Procyanidin B2 alleviates damage to mouse testicular tissue after freezing by inhibiting oxidative stress and apoptosis.","authors":"Guorui Cao, Chunyuan Li, Jian Zhang, Liwen Deng, Rong Li, Changlong Xu","doi":"10.1016/j.cryobiol.2025.105196","DOIUrl":"https://doi.org/10.1016/j.cryobiol.2025.105196","url":null,"abstract":"<p><p>For infertile patients who are unable to obtain sperm or prepubertal boys who require radiotherapy, testicular tissue freezing can be used for later transplantation and is a potentially effective method of preserving male fertility. Oxidative stress caused by the freezing process is an important cause of tissue damage. Procyanidin B2 (PCB2) is a polyphenolic natural compound widely distributed in plants that is known for its anti-inflammatory, anticancer, and neuroprotective properties, and its antioxidant capabilities are particularly noteworthy. Research has indicated that PCB2 exerts a protective effect on the reproductive system. However, its specific role in mitigating testicular tissue cryoinjury and the underlying mechanisms remain unclear. This study investigated whether adding PCB2 to a vitrified cryoprotective solution of mouse testicular tissue can alleviate the cryoinjury of testicular tissue and its possible mechanism. Our findings revealed that frozen mouse testicular tissue presented decreased cell viability and induced oxidative stress. Conversely, PCB2 effectively mitigated these adverse effects. In addition, PCB2 improved the tubular structural disorganization caused by freezing and increased the expression of proteins related to the junction function of Sertoli cells. Further experiments indicated that PCB2 activated the nuclear respiratory factor 2 (Nrf2)/heme oxygenase 1 (HO-1) antioxidant signaling pathway, increased the activity of downstream antioxidant enzymes, and improved mitochondrial kinetic homeostasis. Additionally, PCB2 ameliorated apoptosis while increasing the expression levels of key enzymes involved in testosterone synthesis. In summary, these results suggest that PCB2 attenuates damage to mouse testicular tissue during freezing by inhibiting oxidative stress and apoptosis.</p>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":" ","pages":"105196"},"PeriodicalIF":2.3,"publicationDate":"2025-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142930567","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Detecting changes of testicular interstitial cell membranes with a fluorescent probe after incubation and cryopreservation with cryoprotective agents.","authors":"Oleksandr Pakhomov, Yevgen Posokhov","doi":"10.1016/j.cryobiol.2024.105194","DOIUrl":"https://doi.org/10.1016/j.cryobiol.2024.105194","url":null,"abstract":"<p><p>Membrane alterations are among central factors predetermining cell survival during cryopreservation. In the present research, we tested some serum-/xeno-free cryoprotective compositions including dimethyl sulfoxide (Me₂SO) and polymers for their osmotic impact and toxicity towards testicular interstitial cells (ICs). IC survival was determined after their contact with Me₂SO, dextran (D40), hydroxyethyl starch (HES), polyethylene glycols (PEG1500 and PEG400), or after cryopreservation and cryoprotective agent (CPA) removal. A ratiometric fluorescent membrane probe 2-(2'-hydroxyphenyl)-5-phenyl-1,3-oxazole (probe O1O) was applied to assess changes in the plasma membrane of ICs. The cell survival study has shown that Me₂SO decreased IC survival in a time- and dosage-dependent manner. The CPA decreased the metabolic activity of ICs, thus implying its toxic effect on the living cell as a whole. Using probe O1O, we have demonstrated that the toxic effect also influenced the plasma membrane. IC membranes were not altered after incubation with 0.7 M Me₂SO. The presence of D40, HES, or PEGs in such Me₂SO containing media resulted in plasma membrane hydration and damage to the membranes of cells incubated with PEGs. Cryopreservation caused pronounced membrane dehydration of the survived ICs even after CPA removal in PEG-containing media and low indicators of IC survival. Interestingly, cryopreservation with the best cryoprotective media supplemented with 0.7 M Me₂SO and 100 mg/ml D40 resulted in minimal membrane alterations, thus implying its higher ability to protect membranes during cryopreservation.</p>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":" ","pages":"105194"},"PeriodicalIF":2.3,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142926866","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Antioxidant activity and protective effect of hydroxy derivatives of chalcones for sterlet (Acipenser ruthenus, Linnaeus, 1758) sperm.","authors":"V P Osipova, M N Kolyada, M A Polovinkina, A D Kolumbet, E N Ponomareva, A V Velikorodov","doi":"10.1016/j.cryobiol.2024.105193","DOIUrl":"10.1016/j.cryobiol.2024.105193","url":null,"abstract":"<p><p>The aim of this work is to study the effect of adding hydroxy derivatives of chalcones to the basic cryomedium on the ability of sterlet sperm to utilize superoxide and hydrogen peroxide, the intensity of lipid peroxidation of male fish germ cells, and their viability both before cryopreservation and after 3 days of freezing at liquid nitrogen temperature. The ability of phenolic derivatives of chalcones to increase the superoxide dismutase and catalase activities of sterlet sperm and to reduce the intensity of lipid peroxidation has been established. The antioxidant activity of the derivatives exceeds the effect of Trolox, which inhibits the functioning of the enzyme component of the antioxidant protection of fish sperm and promotes lipid peroxidation of fish sperm before cryopreservation. A beneficial effect of hydroxy derivatives of chalcones on the motility parameters of thawed sperm has been shown, indicating their ability to increase the cryoresistance of sperm in such a promising aquaculture species as sterlet.</p>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":" ","pages":"105193"},"PeriodicalIF":2.3,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142892257","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The role of microRNA in the regulation of hepatic metabolism and energy-expensive processes in the hibernating dormouse.","authors":"W Aline Ingelson-Filpula, Anna Kübber-Heiss, Johanna Painer, Gabrielle Stalder, Hanane Hadj-Moussa, Fabrice Bertile, Caroline Habold, Sylvain Giroud, Kenneth B Storey","doi":"10.1016/j.cryobiol.2024.105191","DOIUrl":"10.1016/j.cryobiol.2024.105191","url":null,"abstract":"<p><p>The garden dormouse (Eliomys quercinus) is a fat-storing mammal that undergoes annual periods of hibernation to mitigate the effects of food scarcity, low ambient temperatures, and reduced photoperiod that characterize winter. Like other hibernating species, this animal suppresses its metabolic rate by downregulating nonessential genes and processes in order to prolong available energy stores and limit waste accumulation throughout the season. MicroRNAs (miRNAs) are short, single-stranded, noncoding RNAs that bind to mRNA and mediate post-transcriptional suppression, making miRNA ideal for modulating widespread changes in gene expression, including global downregulation typified by metabolic rate depression. Using next-generation sequencing, we analyzed an RNA-seq dataset to determine which miRNAs are differentially regulated during hibernation in the dormouse liver. We found that the expression of 19 miRNAs was altered during hibernation; however, only one major miRNA (miR-34a-5p) remained significantly downregulated after correcting for false discovery rate. Gene Ontology, KEGG Pathway Analysis, and DIANA-miRPath predicted that energy metabolism, nuclear-related functions such as histone binding, chromatin- and chromosomal binding, and the cell cycle are processes that may be differentially regulated during hibernation due to miRNA regulation. Taken together, our data suggest that miRNA influence appears to be strongly directed toward suppressing energy-intensive processes in the nucleus hence contributing to extend the animal's endogenous fuel reserves for the duration of hibernation.</p>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":" ","pages":"105191"},"PeriodicalIF":2.3,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142892300","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Curcumin-loaded niosomal nanocarriers offer a promising approach to improve quality characteristics, apoptotic gene expression, and flow cytometry assessments of stallion spermatozoa after thawing.","authors":"Niloofar Nasiri-Foomani, Saeed Hassani, Mojtaba Najafi, Firooz Samadi","doi":"10.1016/j.cryobiol.2024.105188","DOIUrl":"10.1016/j.cryobiol.2024.105188","url":null,"abstract":"<p><p>The optimization of cryopreservation media to reduce oxidative damage on post-thaw spermatozoa is crucial. This research aimed to assess the antioxidant properties of curcumin-loaded niosomal nanocarriers (CurLNN) on the functional characteristics, the relative expression of apoptotic genes, and flow cytometry assessments of apoptotic-like changes, reactive oxygen species production (ROS), mitochondrial membrane potential, and chromatin integrity in stallion spermatozoa following thawing. Twenty-five ejaculates were diluted in INRA96 freezing media supplemented with 20 μM of either curcumin (Cur) or CurLNN and then cryopreserved. Results demonstrated that spermatozoa treated with Cur, particularly CurLNN, exhibited higher percentages of total and progressive motility, as well as VAP, VSL, and STR kinematics. Additionally, the functionality of the plasma membrane was enhanced, and there was a decrease in spermatozoa abnormality (P < 0.05). The incorporation of cryo-diluent medium with Cur and CurLNN led to increased viability (P < 0.05), while simultaneously reducing the levels of MDA. Flow cytometry analysis revealed a significant enhancement in mitochondrial potential activity, a reduction (P < 0.05) in ROS production, and an increase (P < 0.05) in the proportion of live and a decrease in late apoptotic stallion post-thawed spermatozoa treated with both Cur and CurLNN. Moreover, the relative expression of the Bcl₂ anti-apoptotic gene increased (P < 0.05) by the addition of cur and CurLNN in cryo-diluent extender, while inclusion of CurLNN in cryo-diluent medium resulted in a significant reduction (P < 0.05) in the relative expression of the Bax pro-apoptotic gene in stallion post-thawed spermatozoa. In summary, the findings of this study demonstrated that CurLNN exhibits enhanced antioxidant properties, which contribute to the improved functional quality of spermatozoa by alleviating oxidative stress during the cryopreservation process.</p>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":" ","pages":"105188"},"PeriodicalIF":2.3,"publicationDate":"2024-12-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142871626","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"LPA reduces the apoptosis of cryopreserved porcine skin-derived stem cells by inhibiting the regulatory factor TNF-α.","authors":"Xin-Xiang Xie, Jia-Dong Sun, Ming-Xin Zang, Geng Zhang, Chun-Xiao Li, Xiang-Wei Zhai, Wei Shen, Wei Ge, Shun-Feng Cheng","doi":"10.1016/j.cryobiol.2024.105189","DOIUrl":"10.1016/j.cryobiol.2024.105189","url":null,"abstract":"<p><p>Preserving the viability and functionality of stem cells during cryopreservation is crucial for their successful application in regenerative medicine. The aim of this study is to investigate the effect of lysophosphatidic acid (LPA) on reducing the apoptosis of cryopreserved porcine skin-derived stem cells (pSDSCs). Our findings revealed that LPA, at a concentration of 5 μM, significantly improved viability and reduced apoptosis in cryopreserved pSDSCs. Furthermore, our data indicated that LPA enters pSDSCs through receptor type 1 (LPAR1). In cryopreserved pSDSCs, after LPA treatment, the expression level of tumor necrosis factor alpha (TNF-α) protein decreased, suggesting TNF-α involvement in the regulation of the anti-apoptotic process. Additionally, we found that resiquimod (R848), a TNF-α activator, increased the level of apoptosis in cryopreserved pSDSCs. When cryopreserved pSDSCs were treated with both LPA and R848, the protective effect of LPA against apoptosis was decreased. In conclusion, our study demonstrates that LPA could effectively counteract the effect of TNF-α-induced apoptosis, thereby enhancing the survival rates of cryopreserved pSDSCs. Importantly, this study explored a novel mechanism of reducing apoptosis in cryopreserved stem cells.</p>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":" ","pages":"105189"},"PeriodicalIF":2.3,"publicationDate":"2024-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142871627","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}